Mitochondria are functionally diverse organelles with a central role in many cellular processes, including oxidative phosphorylation and apoptosis. The mitochondrial theory of aging postulates that the lifelong accumulation of mitochondrial DNA (mtDNA) mutations in multiple tissues leads to mitochondrial failure, downstream processes such as apoptosis, and the progressive decline of tissue function (i.e., aging and disease). Moreover, mtDNA mutations are suspected to contribute to the etiology of a number of age-related disorders, including Parkinson's disease, muscle-wasting, and the metastatic potential of cancers. As eukaryotic cells contain many hundreds copies of mtDNA, it stands to reason that a minimum critical number of mutant mtDNAs must be present before tissue dysfunction and clinical signs become apparent. Yet, we do not fully understand the mechanisms underlying the amplification and fixation of these pathogenic mutant populations. There is evidence that these mutations pre-exist at extremely low frequency as random mutations in normal cells and tissues. Our working hypothesis is that the induction and accumulation of random mtDNA mutations fuels pathogenic mutant populations, aging, and age-related disease. Thus, environmental mutagens in particular may play a substantial role in the origin and incidence of aging and disease by damaging mtDNA and increasing the rate at which mitochondrial mutations accumulate. The goal of this proposal is to determine the molecular mechanisms of somatic mtDNA mutagenesis associated with DNA damaging agents and disease, under the direction of the following Aims: (1) test the hypothesis that the frequency of random mtDNA mutations increases with age in humans, (2) determine whether an environmental toxin can increase the rate at which mitochondrial mutations accumulate, (3) evaluate whether random mtDNA mutations precede and drive pathogenic mutant populations in human cells, and (4) reduce mtDNA mutations in human cells. Our Specific Aims are feasible, because of our dramatic advance in our ability to sensitively measure mitochondrial mutations, and important, given the critical role of mitochondria in aging and cancer. A mechanistic understanding of the environmental factors that accelerate mtDNA mutation should aid in the identification of risk factors and methods that prevent and/or slow age-related debilitation and disease. Ultimately, fulfillment of our proposed aims will help unravel the role of mitochondrial mutagenesis in human aging and potentially aid in the amelioration of age-related disease, extending the number of healthy and active years of life. PUBLIC HEALTH RELEVANCE: Mitochondrial mutations are suspected to contribute to the etiology of a number of age-related disorders, including Parkinson's disease, muscle-wasting, and the metastatic potential of cancers. Yet, we do not fully understand the mechanisms underlying the generation of mitochondrial mutations. Ultimately, fulfillment of our proposed aims, to elucidate mitochondrial mutagenesis, will aid in the amelioration of age-related disease, and extend the number of healthy active years of life.